Variola virus (Smallpox) is a Category A pathogen considered to be one of the most significant threats for use as a bioterrorism agent. Due to complications from vaccination, mass immunization of the populace is contra-indicated. Our current research seeks to develop effective anti-orthopoxvirus drug(s), which is designated as a high priority biodefense project. Using vaccinia virus (VV) as a model system, the goal of our previous research was to determine if the l7L cysteine proteinase or the G1L metalloproteinase encoded by W is the poxvirus core protein proteinase (vCPP), and to use this information to develop vCPP inhibitors as candidate antiviral drugs. We have recently demonstrated that the l7L cysteine proteinase is the vCPP and are proceeding with drug development efforts on this target. But what about G1L? This represents an unexpected opportunity that should be investigated. We believe that the W G1L metalloproteinase represents a unique and distinct orthopoxvirus antiviral target. The purpose of the experiments outlined in this application are to: 1) Produce a G1L conditional-lethal mutant to assess the phenotype of the null mutant; 2) Elucidate the biological role of G1L during viral replication and/or assembly; and 3) Demonstrate and characterize the enzymatic activity of the G1L gene product. Successful completion of these experiments should allow the development of G1L metalloproteinase inhibitors as antiviral drugs to be initiated. There are several important reasons to exploit the G1L target in addition to l7L: Not all enzymes are equally "drug-able" and G1L inhibitors may have superior activity/specificity profiles; When exposed to selective pressure, viruses rapidly acquire resistance so having multiple antiviral drugs available is essential; and using a cocktail approach with multiple inhibitors may be more effective than using a single drug.